The overall objective of this project is to develop a prostate cancer diagnostic test for biopsies with atypical findings. Over one million prostate biopsies are performed in the US each year. Approximately 10% show abnormalities that are suspicious but not diagnostic of cancer. These include high-grade prostatic intraepithelial neoplasia (HGPIN) and atypical small acinar proliferation (ASAP). Abnormal biopsies are a strong indicator of subsequent diagnosis of cancer. Our aim is to develop a diagnostic test based on DNA methylation to identify patients with abnormal biopsies who are at higher risk of a cancer diagnosis upon repeat biopsy. This test should help reduce the number of unnecessary repeat biopsies. Towards this objective, we have identified 12 novel markers that are methylated in prostate cancer. We have also developed an improved method to simultaneously detect the methylation pattern and density within a CpG island using termination-coupled linear amplification (TCLA). A PCA test based on the analysis of methylated markers using TCLA should reduce the number of repeat biopsies by at least 50% and is commercially valuable as an additional tool to improve prostate cancer diagnosis. In this application, we propose to apply TCLA to detect the methylation patterns of the new markers in DNA isolated from abnormal biopsies and correlate the level of methylation to the risk of positive diagnosis on repeat biopsies. If successful, we will undertake a large validation study during Phase II in preparation for a clinical trial. PUBLIC HEALTH RELEVANCE: Over one million prostate biopsies are performed each year in the US due to elevated PSA levels leading to the diagnosis of about 180,000 cases of prostate cancer. Approximately 10% of biopsies find abnormal cells that are indicative of cancer. The aim of this study is to develop a prostate cancer diagnostic test that can identify patients with abnormal biopsies who are at higher risk of prostate cancer diagnosis and would benefit from a repeat biopsy.